RESUMEN
BACKGROUND: Probiotics are widely studied both in the treatment and prevention of allergic diseases, but their mode of action is poorly known. OBJECTIVE: Our aim was to examine the effect of probiotic bacteria on in vivo cytokine, antibody, and inflammatory responses in allergy-prone infants. METHODS: In a randomized double-blind study, probiotic bacteria or placebo were given for 1 month before delivery to mothers and for 6 months to infants with a family history of allergy. Plasma samples were analysed for C-reactive protein (CRP), total IgA and IgE, food-specific IgA, IgG, and IgE, IL-2, IL-4, IL-6, IL-10, TNF-alpha, and IFN-gamma. We analysed the associations of immunological and inflammatory parameters at age 6 months with probiotic treatment and allergic phenotype at 2 years. RESULTS: Infants receiving probiotic bacteria had higher plasma levels of CRP (P=0.008), total IgA (P=0.016), total IgE (P=0.047), and IL-10 (P=0.002) than infants in the placebo group. Increased plasma CRP level at age 6 months was associated with a decreased risk of eczema [odds ratio (OR) 0.41 [95% confidence interval (CI) 0.17-0.99], P=0.046], and with a decreased risk of allergic disease [OR 0.38 (95% CI 0.16-0.87), P=0.023] at age 2 years, when adjusted with probiotic use. CONCLUSION: The association of CRP with a decreased risk of eczema at 2 years of age in allergy-prone children supports the view that chronic, low-grade inflammation protects from eczema. Probiotic-induced low-grade inflammation was characterized by elevation of IgE, IgA, and IL-10, the changes typically observed in helminth infection-associated induction of regulatory mechanisms. The findings emphasize the role of chronic microbial exposure as an immune modulator protecting from allergy.
Asunto(s)
Eccema/inmunología , Eccema/prevención & control , Hipersensibilidad/inmunología , Hipersensibilidad/prevención & control , Inflamación/inmunología , Probióticos/administración & dosificación , Proteína C-Reactiva/análisis , Preescolar , Enfermedad Crónica , Citocinas/sangre , Método Doble Ciego , Eccema/diagnóstico , Femenino , Estudios de Seguimiento , Humanos , Hipersensibilidad/terapia , Inmunoglobulina A/sangre , Inmunoglobulina E/sangre , Lactante , Oportunidad Relativa , Fenotipo , Placebos , Valor Predictivo de las Pruebas , Embarazo , Factores de Riesgo , Sensibilidad y Especificidad , Pruebas CutáneasRESUMEN
BACKGROUND: The prevalence of lactase persistence is high in Saudi Arabia. OBJECTIVE: To identify a DNA variant for the lactase persistence/non-persistence trait in adult Arabs in Saudi Arabia. METHODS: We sequenced DNA from 432 anonymous neonatal blood donors from five different regions of Saudi Arabia to cover the 400 bp region surrounding the previously identified lactase persistence/non-persistence variant C/T-13910 residing in intron 13 of the MCM6 gene. RESULTS: Two anonymous blood donors carried the C/T-13910 genotype. One variant, T/G -13915, residing 5 bp upstream of the C/T-13910 variant, was present in 332 of 432 (76.9%) of the neonatal samples, compatible with previous prevalence figures of lactase persistence in urban Saudi populations. Determination of disaccharidase activities in 25 intestinal biopsy samples showed a highly significant correlation between lactase activity and the T/G-13915 genotypes (p<0.001; Fisher exact test) as well as between the L:S ratio and the aforementioned genotypes (p<0.001; Fisher exact test). CONCLUSION: The T/G-13915 variant is the founder mutation of lactase persistence in an urban Saudi population. The results obtained here have implications for genetic testing of adult-type hypolactasia and to analysis of human evolution, the origin of cattle domestication and migrations of the populations in the Arabian peninsula.
Asunto(s)
Regulación de la Expresión Génica , Variación Genética , Lactasa/genética , Lactatos/metabolismo , Alelos , Biopsia , Evolución Molecular , Efecto Fundador , Genotipo , Humanos , Recién Nacido , Intrones , Lactasa/fisiología , Modelos Genéticos , Arabia Saudita , Población UrbanaRESUMEN
Eight human myeloma proteins, two of each IgG subclass, were studied for binding to solid-phase C3c and C3d by the ELISA technique. Myeloma IgG1 kappa, IgG1 lambda, IgG3 kappa and IgG3 lambda proteins bound to C3c and C3d, while two IgG2 kappa, and two IgG4 kappa proteins failed to show significant binding affinity. The results suggest that like C1q, the stable binding sites of C3, located on the C3c and C3d parts of the molecule, have affinity for IgG subclasses 1 and 3.
Asunto(s)
Complemento C3/metabolismo , Inmunoglobulina G/metabolismo , Proteínas de Mieloma/metabolismo , Receptores de Complemento , Animales , Unión Competitiva , Fenómenos Químicos , Química , Complemento C3c , Complemento C3d , Ensayo de Inmunoadsorción Enzimática , Humanos , ConejosRESUMEN
Proteins that decrease the surface activity of surfactant accumulate in epithelial lining fluid in respiratory failure. The aim of this study was to isolate a surfactant inhibitor from the airways of rabbits in acute respiratory failure induced by bronchoalveolar lavage (BAL). This inhibitor was identified as being transferrin (TF). Unlike serum TF, TF recovered in respiratory failure was saturated with iron (Fe(3+)-TF). Fe(3+)-TF decreased the surface activity of normal surfactant in vitro, whereas iron-free TF had no effect. In the presence of H2O2 and a reducing agent, Fe(+3)-TF inactivated the surfactant complex: the surface absorption rate was decreased, immunoreactive surfactant protein A was decreased, and malondialdehyde was formed. The acute effects of Fe(3+)-TF and iron-free TF applied to the airways were studied in animal models. In respiratory failure induced by BAL, Fe(3+)-TF deteriorated respiratory failure, whereas iron-free TF had no effect. In respiratory failure induced by hyperoxia for 48 h, administration of iron-free TF ameliorated the respiratory failure and improved the surface activity in BAL. We propose that Fe(3+)-TF accumulating in epithelial lining fluid during lung damage contributes to surfactant inhibition and promotes the formation of free radicals that inactivate the surfactant system.
Asunto(s)
Hierro/metabolismo , Surfactantes Pulmonares/metabolismo , Insuficiencia Respiratoria/metabolismo , Transferrina/metabolismo , Animales , Líquido del Lavado Bronquioalveolar/citología , Electroforesis en Gel de Poliacrilamida , Radicales Libres/metabolismo , Glicoproteínas/inmunología , Glicoproteínas/metabolismo , Malondialdehído/metabolismo , Peso Molecular , Consumo de Oxígeno , Proteolípidos/inmunología , Proteolípidos/metabolismo , Edema Pulmonar/complicaciones , Edema Pulmonar/fisiopatología , Proteínas Asociadas a Surfactante Pulmonar , Surfactantes Pulmonares/antagonistas & inhibidores , Surfactantes Pulmonares/deficiencia , Surfactantes Pulmonares/inmunología , Conejos , Pruebas de Función Respiratoria , Insuficiencia Respiratoria/etiología , Insuficiencia Respiratoria/fisiopatologíaRESUMEN
Mice were immunized with purified human IgG myeloma proteins and hybridomas were prepared using their spleen cells. 1817 of the hybridomas secreted anti-Ig antibodies. Several of them detected subclass-associated determinants. Eight different specificities could be distinguished. The number of hybridomas in each category were the following: 3 anti-IgG1 (1.8% of all anti-Ig clones) 5 anti-IgG2 (0.3% of all anti-Ig clones) 2 anti-IgG3 (2.5% of all anti-Ig clones) 3 anti-IgG4 (12% of all anti-Ig clones) 12 anti-IgG1, IgG2, IgG3 70 anti-IgG1, IgG2, IgG4 2 anti-IgG2, IgG4 7 anti-IgG2, IgG4 (one out of five myelomas).
Asunto(s)
Anticuerpos Monoclonales/inmunología , Inmunoglobulina G/análisis , Especificidad de Anticuerpos , Epítopos/análisis , Humanos , Hibridomas/inmunología , Alotipos de Inmunoglobulinas/análisis , Inmunoglobulina G/clasificación , Proteínas de Mieloma/inmunologíaRESUMEN
In the present study, the expression of type IV collagen associated with the basal membrane (BM) was studied histochemically (indirect immunoperoxidase-antiperoxidase) in cervical human papillomavirus (HPV) lesions (diagnosed using in situ DNA hybridization) of different grades. The expression of type IV collagen in premalignant epithelial lesions (HPV with and without cervical intraepithelial neoplasia) was identical with that in the BM of normal exocervical epithelium, in contrast to 60% (3/5) of carcinoma in situ lesions and 90% (10/11) of invasive carcinomas, where the staining pattern was interrupted and the staining intensity reduced. Thus, the expression of type IV collagen seems to remain unchanged during the entire spectrum of premalignant stages of cervical HPV lesions. This suggests that the squamous epithelial cells responsible for the formation of the BM are not affected by this virus at early stages of the disease, and immunohistochemical recognition of an intact staining pattern of type IV collagen may signify well-preserved basal cell function (confined to nonmalignant?) HPV-infected squamous epithelium.
Asunto(s)
Membrana Basal/análisis , Carcinoma de Células Escamosas/análisis , Colágeno/análisis , Lesiones Precancerosas/análisis , Infecciones Tumorales por Virus , Neoplasias del Cuello Uterino/análisis , Membrana Basal/metabolismo , Membrana Basal/patología , Carcinoma de Células Escamosas/patología , Femenino , Humanos , Técnicas para Inmunoenzimas , Papillomaviridae , Lesiones Precancerosas/patología , Estudios Prospectivos , Neoplasias del Cuello Uterino/patologíaRESUMEN
Mumps antibodies of 34 human beings were studied, 12 patients with natural mumps infection, 15 subjects vaccinated with a live mumps vaccine, and seven subjects vaccinated with an inactivated vaccine. Small amounts of antibodies reacting with mumps antigen were found in the prevaccination sera. An immunization with either the live or the killed vaccine caused an increase in the mumps antibodies (range, from 1.1-fold to more than 50-fold; geometric mean, approximately sevenfold). IgG1 was the major isotype in all post-vaccination sera; the average share was 61%. Next came IgM (28%), followed by IgA (9%), and IgG3 (2% of total). The patient samples had 10 (acute phase) or 20 times (convalescent phase) more mumps antibodies than the prevaccination samples. IgG1 was the predominant isotype in the acute phase sera (average 42% of all antibodies). Next came IgM (41%) followed by IgA (13%), and IgG3 (4%). In convalescent sera IgG1 was also the predominant isotype (average 67%), followed by IgM (19%). The minor isotypes in the second samples were IgA (12%) and IgG3 (3%). Small amounts of IgG2 antibodies were found in 1 patient and 1 vaccine. IgG4 antibodies were not detected.
Asunto(s)
Anticuerpos Antivirales/análisis , Inmunoglobulina G/clasificación , Virus de la Parotiditis/inmunología , Humanos , Inmunoglobulina A/análisis , Inmunoglobulina G/análisis , Inmunoglobulina M/análisis , Vacuna contra la Parotiditis/administración & dosificación , Vacuna contra la Parotiditis/inmunologíaRESUMEN
We investigated gut permeability to human alpha-lactalbumin (ALA) and bovine beta-lactoglobulin (BLG) in 20 infants from birth to 8 months or until weaning, before which they were on a strictly cow's-milk-free diet. We measured the proteins with a sensitive, solid-phase, double-sandwich immunofluorometric assay. Median (range) levels of serum ALA on days 3-4 after birth, and at 1 and 2 months of age were 31 (12-225), 6 (0-55), and 2 (0-16) micrograms/l serum per g ALA given per kg body weight, respectively. At 3, 5, and 8 months of age, only trace amounts of ALA were found. One week after weaning, serum BLG was found in 5/13 infants (38%) and at 2 weeks in 3/14 infants (21%), with median concentrations of 7 and 4 micrograms/l serum per g BLG given per kg body weight, respectively. No ALA could be detected in any of these samples. In absorption of ALA, the four infants who had allergic symptoms did not differ from those without symptoms. Thus, systemic absorption of ALA and BLG does occur in infants. Absorption of ALA is greatest after birth, when 3 x 10(-4) (median) of the given antigens are absorbed, but absorption decreases rapidly. The gut may often be transiently permeable to BLG when cow's-milk-based formula is started.
Asunto(s)
Inmunoglobulina A Secretora/biosíntesis , Inmunoglobulina G/metabolismo , Absorción Intestinal , Lactalbúmina/farmacocinética , Lactoglobulinas/farmacocinética , Hipersensibilidad a la Leche/sangre , Leche Humana/metabolismo , Leche/metabolismo , Factores de Edad , Animales , Peso Corporal , Lactancia Materna , Bovinos , Heces/química , Fluoroinmunoensayo , Humanos , Inmunoglobulina A Secretora/sangre , Inmunoglobulina A Secretora/inmunología , Lactante , Alimentos Infantiles/análisis , Recién Nacido , Lactalbúmina/sangre , Lactoglobulinas/sangre , Leche/inmunología , Hipersensibilidad a la Leche/inmunología , Leche Humana/inmunología , Permeabilidad , Saliva/metabolismo , Factores de TiempoRESUMEN
The proportions of six immunoglobulin isotypes (IgA, IgM, IgG1, IgG2, IgG3, and IgG4) in rubella antibody responses were quantified in 40 serum samples (20 patients). The first sample from each patient was taken during the first days of the illness, and the second sample 10 +/- 1 days later. A tenfold average increase in antibody concentration was observed between the first and the second sample. IgM was the predominant isotype in the first sample (average, 73% of all antibodies), followed by IgG1 (19%). IgA and IgG3 antibodies were detected in only a few of the first samples, and IgG2 or IgG4 in none. In the second samples IgG1 was the predominant antibody isotype (average, 59%). Next came IgM (23%), followed by IgA (8%) and IgG3 (3%). No IgG2 or IgG4 antibodies were detected. Although the proportion of IgM antibodies was lower in the second than in the first samples, their concentration increased in all patients (the average factor was 7). The kinetics of the IgA response was irregular. In some patients there was a strong (up to 90-fold) increase in IgA antibodies, but in two patients a small drop was detected. The kappa- to lambda-chain ratio of rubella antibodies appears to be close to the expected 2:1. It decreased in some patients during the 10 days and increased in others.
Asunto(s)
Anticuerpos Antivirales/análisis , Inmunoglobulinas/análisis , Virus de la Rubéola/inmunología , Humanos , Inmunoglobulina A/análisis , Inmunoglobulina G/análisis , Inmunoglobulina M/análisis , Cadenas alfa de Inmunoglobulina/análisis , Cadenas kappa de Inmunoglobulina/análisis , Cinética , Factores de TiempoRESUMEN
The human serum enzyme, beta-galactoside alpha 1----2 fucosyltransferase, presumably blood group H gene-encoded, was purified to homogeneity from serum of AB and mixed secretor phenotype individuals. The purification procedure involved chromatography on phenyl-Sepharose, S-Sepharose, GDP-hexanolamine-Sepharose, and high pressure liquid chromatography gel filtration. The enzyme was purified 10 x 10(6)-fold, with a final specific activity of 23.6 units/mg for the phenyl-beta-O-galactoside acceptor. The apparent Mr of the H gene-encoded beta-galactoside alpha 1----2 fucosyltransferase was determined as 200,000 and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in nonreducing and reducing conditions, respectively. The Mr of native enzyme was found by gel filtration chromatography to be 148,000. The subunit structure as well as the sensitivity of the enzymatic activity to beta-mercaptoethanol suggest that the native enzyme exists in polymeric form of covalently bound subunits. Lectin binding properties of the purified molecule indicate that the enzyme is glycosylated. Another human serum beta-galactoside alpha 1----2 fucosyltransferase, presumably Se gene-encoded, was separated from the H enzyme by adsorption on S-Sepharose cation exchange matrix. A comparison of the kinetic parameters of the initial rate data of both alpha 1----2 fucosyltransferases revealed differences between Km values for various oligosaccharide acceptors. Higher Km values for the phenyl-beta-O-galactoside acceptor and a lower Km for the lacto-N-tetraose-beta-O-PA8 type 1 acceptor for the enzyme that adsorbed to S-Sepharose compared with nonadsorbed enzyme were observed. The two enzymes also were differentiated by binding properties to S-Sepharose and electrophoretic mobilities on native gel electrophoresis. We, therefore, postulate that the enzyme which does not adsorb to S-Sepharose and adsorbed enzyme are structurally different molecules and they represent the H and Se gene-encoded beta-galactoside alpha 1----2 fucosyltransferases, respectively.
Asunto(s)
Antígenos de Grupos Sanguíneos/genética , Fucosiltransferasas/sangre , Hexosiltransferasas/sangre , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Fucosiltransferasas/genética , Fucosiltransferasas/aislamiento & purificación , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Peso Molecular , Especificidad por Sustrato , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
The absorption of alpha-lactalbumin (ALA) and bovine beta-lactoglobulin (BLG) was investigated in 23 healthy preterm infants with gestational ages of 32 to 36 wk. The concentrations of ALA and BLG in serum after a milk feeding were measured at intervals during the first 8 mo of life. We used a time-resolved fluoroimmunoassay to measure the proteins. Measurable amounts of ALA were found on d 7 after birth, and at 1, 2, 3, 5, and 8 mo in 23 of 23, 13 of 18, 13 of 18, six of 17, eight of 16, and five of 13, respectively, of the infants tested; median serum levels of ALA at the respective ages were 120 (range, 19-2598), 16 (range, 0-177), 5 (range, 0-40), 0 (range 0-3), 0.8 (range 0-38), and 0 (range, 0-22) micrograms/L serum/g ALA given/kg body wt, respectively. The rate of decline in ALA absorption was comparable among the infants. Tests for BLG were begun after the introduction of cow's milk. At 2, 3, 5, and 8 mo of age BLG was detected in two of 7, two of 9, eight of 10, and two of 12, respectively, of the infants tested, where median levels in positive cases were 13, 17, 15, and 3 micrograms/L serum/g BLG given/kg, respectively. The amounts of absorbed ALA and BLG were 10(-5) to 10(-3) of the oral dose. Serum levels of ALA or BLG did not depend on the gestational age of the infant. Few of the infants had any detectable absorption of either protein shortly after weaning.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Recien Nacido Prematuro/metabolismo , Lactalbúmina/farmacocinética , Lactoglobulinas/farmacocinética , Factores de Edad , Animales , Antígenos/metabolismo , Bovinos , Humanos , Lactante , Recién Nacido , Recien Nacido Prematuro/inmunología , Absorción Intestinal , Mucosa Intestinal/metabolismo , Intestinos/inmunología , Lactalbúmina/inmunología , Lactoglobulinas/inmunología , Leche/inmunología , Leche/metabolismo , Leche Humana/inmunología , Leche Humana/metabolismo , PermeabilidadRESUMEN
Xanthine dehydrogenase/xanthine oxidase (XDH/XO) is a major cytoplasmic source of superoxide radicals and hydrogen peroxide, and it is considered important in the pathogenesis of ischemia-reperfusion damage. Because little is known about the enzyme in human tissues, the aims of this study were to purify human XDH/XO and to produce Ab for detection of the protein in Western blots and for quantification by ELISA. We purified human milk XDH/XO, produced Ab for Western blotting and ELISA of the protein, and evaluated the molecular forms and activity-protein relationships in human tissues. The molecular size of the purified protein under nondenaturing conditions was approximately 300 kd. On SDS-PAGE, it was fragmented into four main bands of 143, 125, 87, and 59 kd. Ab recognized bands of similar size in Western blots of the purified preparation and human milk. In fresh liver homogenates treated with anti-proteases, the three largest bands were observed; in the intestine, only the two largest were observed. Serum, brain, heart, and skeletal muscle were negative, whereas some lung and kidney samples showed one faint band of 143 kd. Trypsin treatment of the enzyme converted the large molecular-weight bands into smaller bands, as did incubation of a liver homogenate without anti-proteases. XDH/XO protein concentrations (ng/mg total protein) were 146 +/- 70 in liver and 556 +/- 320 in intestine and less than 5 ng/ml in serum. The relationship of activity to protein (2.7-3.0 mumol/min/mg XDH/XO protein) was constant in liver and intestine during development. We conclude that 1) human XDH/XO has molecular size and subunit structure similar to other mammalian enzymes; 2) the polypeptide chain is unstable, also in the intact cell, despite retained activity; and 3) the amount of inactive XDH/XO in human liver and intestine is apparently small.
Asunto(s)
Especificidad de Órganos , Xantina Deshidrogenasa/química , Xantina Oxidasa/química , Adulto , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Endopeptidasas , Sangre Fetal/enzimología , Feto , Humanos , Hidrólisis , Sueros Inmunes/química , Recién Nacido , Leche Humana/enzimología , Datos de Secuencia Molecular , Relación Estructura-Actividad , Xantina Deshidrogenasa/sangre , Xantina Deshidrogenasa/aislamiento & purificación , Xantina Oxidasa/sangre , Xantina Oxidasa/aislamiento & purificaciónRESUMEN
Monoclonal mouse antibodies to human IgG myeloma proteins were produced and characterized by determining their binding to a series of different purified myeloma proteins. Two types of immunization schedules were used. When the same myeloma protein was used for priming and boosting the mouse, all determinants of the molecule were effectively immunogenic. Of the 353 clones originating from these experiments 42% secreted anti-Fv antibodies, 57% anti-CH antibodies, and 0.85% anti-CL antibodies. In another schedule only the CL and CH regions were the same in priming and boosting; 270 anti-CH (94%), 18 anti-CL (16%), and no anti-FV hybridomas were found. Our results indicate that a unit mass of the FV region was 1.3 times more antigenic than a unit mass of the CH region. A unit mass of the CH region was nearly five times more antigenic than a unit mass of the CL domain when the antigenicity of the FV region had been excluded. When the antigenicity of the FV region had not been excluded, a unit mass of the CH region was about twenty times more antigenic than a unit mass of the CL domain. The data suggest that an antigenic competition was taking place between different parts of the molecule and that the strongly antigenic region (FV) was more efficient in competing out the nearest neighbour (CL) than in competing out other parts of the molecule.
Asunto(s)
Inmunoglobulina G/inmunología , Proteínas de Mieloma/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Bordetella pertussis/inmunología , Epítopos/análisis , Humanos , Hibridomas , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/inmunología , Ratones , Ratones Endogámicos BALB CRESUMEN
The biosynthetic pathways for the difucosylated type 1 and 2 glycolipids, Leb and Y, respectively, were investigated in the gastric carcinoma cell line KATO III, using a novel chromatogram binding assay. The type of fucosylation obtained was deduced from the binding pattern of monoclonal antibodies specific for the biosynthesized glycolipid products using microsomal fractions as the source of enzyme, pure glycolipids and non-radioactive GDP-fucose as acceptor and donor substrates, respectively. The Leb glycolipid (Fuc alpha 1----2Gal beta 1----3GlcNAc(4----1 alpha Fuc) beta 1----3LacCer) was synthesized mainly via the blood group H, type 1, precursor (Fuc alpha 1----2Gal beta 1----3GlcNAc beta 1----3LacCer). However, the Lea glycolipid (Gal beta 1----3GlcNAc(4----1 alpha Fuc)beta 1----3LacCer) also served as a precursor for the alpha 1----2 fucosyltransferase, thus allowing conversion of Lea to Leb. This biosynthetic route represents either an "aberrant" specificity of the Fuc alpha 1----2 transferase associated with these gastric carcinoma cells and/or a new member of the alpha 1----2 fucosyltransferase family. The Y glycolipid (Fuc alpha 1----2Gal beta 1----4GlcNAc(3----1 alpha Fuc)beta 1----3LacCer) was synthesized exclusively via the classical pathway using the blood group H type 2 glycolipid (Fuc alpha 1----2Gal beta 1----4GlcNAc beta 1----3LacCer) as precursor. The X glycolipid (Gal beta 1----4GlcNAc(3----1 alpha Fuc)beta 1----3LacCer) did not serve as an acceptor substrate for the alpha 1----2 fucosyltransferase(s) present. The use of non-radioactive sugar-nucleotides as donor substrate, defined glycolipid precursors as acceptor substrates and of specific monoclonal anti-glycolipid antibodies for detection provides a rapid and highly specific assay for analyzing biosynthetic pathways of glycosyltransferases.
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Fucosiltransferasas/metabolismo , Glicoesfingolípidos/biosíntesis , Hexosiltransferasas/metabolismo , Neoplasias Gástricas/metabolismo , Antígenos , Autorradiografía , Cromatografía en Capa Delgada , Humanos , Especificidad por Sustrato , Células Tumorales Cultivadas , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
The secretor-type beta-galactoside alpha 1----2-fucosyltransferase from human serum was purified by hydrophobic chromatography on phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, and affinity chromatography on GDP-hexanolamine-Sepharose. Final purification of the enzyme was achieved by high pressure liquid chromatography gel filtration and resulted in a homogeneous protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the radiolabeled protein. The native enzyme appears as a molecule of apparent Mr 150,000 as determined by gel filtration high pressure liquid chromatography. The apparent Mr of the enzyme resolved in the presence of beta-mercaptoethanol by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined to be 50,000, indicating a multisubunit structure of the enzyme. Secretor-type alpha 1----2-fucosyltransferase is a glycoprotein as determined by WGA binding properties. A comparison of the Mr of the native blood group H gene encoded with the secretor-type beta-galactoside alpha 1----2-fucosyltransferases as well as comparison of subunit Mr for both enzymes suggests structural similarity. The alpha 1----2 linkage formed between alpha-L-fucose and terminal beta-D-galactose by the purified H- and secretor-type alpha 1----2-fucosyltransferases was determined by 1H NMR homonuclear cross-irradiation analysis of the oligosaccharide products. The substrate specificity and Km values calculated from the initial rate using various oligosaccharide acceptors showed that purified enzymes differ primarily in affinity for phenyl-beta-D-galactopyranoside and GDP-fucose as well as type 1 (Gal beta 1----3GlcNAc), 2 (Gal beta 1----4GlcNAc), and 3 (Gal beta 1----3GalNAc) oligosaccharide acceptors. The secretor-type alpha 1----2-fucosyltransferase shows significantly lower affinity than the H enzyme for phenyl-beta-D-galactopyranoside and GDP-fucose as well as for type 2 oligosaccharide acceptors. On the contrary, type 1 and 3 oligosaccharide acceptors are preferentially utilized by the secretor-type enzyme as compared with the H enzyme. The enzymes also differ in several physicochemical properties, implying nonidentity of the two enzymes (Sarnesto, A., Köhlin, T., Thurin, J., and Blaszczyk-Thurin, M. (1990) J. Biol. Chem. 265, 15067-15075).
Asunto(s)
Fucosiltransferasas/aislamiento & purificación , Secuencia de Carbohidratos , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Fucosiltransferasas/sangre , Humanos , Espectroscopía de Resonancia Magnética , Mercaptoetanol/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Oligosacáridos/metabolismo , Especificidad por Sustrato , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
A beta-N-Acetylglucosaminide alpha 1----3-fucosyltransferase was purified from human serum by ammonium sulfate precipitation, hydrophobic chromatography on phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, affinity chromatography on GDP-hexanolamine-Sepharose, and finally high pressure liquid chromatography gel filtration. Gel filtration chromatography of the native enzyme revealed a Mr of 45,000. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified protein also appeared as a single molecular species of Mr 45,000. In contrast to the multisubunit beta-galactoside alpha 1----2-fucosyltransferases with an apparent Mr of 150,000, present in human serum, the native beta-N-acetylglucosaminide alpha 1----3-fucosyltransferase is a monomer with a Mr of 45,000. The enzyme is glycosylated, as revealed by wheat germ agglutinin binding properties. The alpha 1----3 linkage formed by the enzyme between alpha-L-fucose and the penultimate beta-N-acetylglucosamine by the purified enzyme was confirmed by 1H NMR homonuclear cross-irradiation analysis of the oligosaccharide product. The specificity of the purified enzyme is restricted to type 2 structures, as revealed by its reactivity with different substrates and from the Km values calculated from the initial rate data using various oligosaccharide acceptors. The enzyme has the ability to utilize the N-acetyl-beta-lactosamine determinant (Gal beta 1----4GlcNAc) and the sialylated (NeuAc alpha 2----3Gal beta 1----4GlcNAc) and fucosylated (Fuc alpha 1----2Gal beta 1----4GlcNAc) derivatives of N-acetyl-beta-lactosamine and thus is distinct from both the human Lewis gene-encoded enzyme and the alpha 1----3-fucosyltransferase of the myeloid cell type.
Asunto(s)
Fucosiltransferasas/aislamiento & purificación , Secuencia de Carbohidratos , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Fucosiltransferasas/sangre , Glicosilación , Humanos , Espectroscopía de Resonancia Magnética , Mercaptoetanol/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Oligosacáridos/metabolismoRESUMEN
Oxygen free radical generation by xanthine oxidase (XO) is a possible mechanism in the injury following reperfusion of transplanted organs. This study was undertaken to investigate XO in human lung, and to investigate whether XO is released into the blood stream during the immediate postoperative period after lung transplantation. XO activity was measured in healthy human lung tissue, and XO protein and the adenine nucleotide catabolic products hypoxanthine, xanthine and uric acid were analysed in the plasma samples collected during human heart-lung transplantation (n=4), double lung transplantation (n=2), and single lung transplantation (n=1). Neutrophil degranulation was assessed by plasma lactoferrin measurements. The results indicated that XO activity (detection limit 5 pmol x min(-1) x mg(-1) protein) and protein (detection limit 5 ng x mg-1 protein) were undetectable in the lungs of five healthy individuals. Similarly, no XO protein could be found in the plasma samples from the right ventricle or left atrium during and after the transplantation in any of the cases. Plasma xanthine and hypoxanthine concentrations were elevated 2-10 fold immediately after the reperfusion of the transplant, indicating washout of high-energy phosphate degradation products from the ischaemic lung. Plasma uric acid decreased rather than increased immediately after the surgery and during the following 24 h. Lactoferrin was elevated during the surgery. In conclusion, these results show that XO activity in human lung is low, it is not released into the blood stream during human heart-lung transplantation, and it is unlikely to contribute to postoperative complications in these patients.
Asunto(s)
Trasplante de Corazón-Pulmón , Trasplante de Pulmón , Pulmón/enzimología , Complicaciones Posoperatorias/enzimología , Daño por Reperfusión/enzimología , Xantina Oxidasa/metabolismo , Cromatografía Líquida de Alta Presión , Ensayo de Inmunoadsorción Enzimática , Radicales Libres , Humanos , Hipoxantina/sangre , Lactoferrina/sangre , Persona de Mediana Edad , Complicaciones Posoperatorias/etiología , Daño por Reperfusión/etiología , Ácido Úrico/sangre , Xantina , Xantina Oxidasa/sangre , Xantinas/sangreRESUMEN
The solid-phase radioimmunoassay for human antibodies was improved so that it gave the total concentration as well as the concentrations of IgM, IgA, IgG1, IgG2, IgG3 and IgG4 antibodies, all seven in the same "L units"/ml. This was accomplished by standardization of monoclonal antibodies with monoisotypic antibodies rather than myelomas; myelomas were found unsatisfactory for this purpose. For the first time it was possible to determine the isotype composition of an antibody population in percent terms. The weight equivalent of the L-unit in one hyperimmune tetanus antitoxin preparation was approximately 7.4 ng. The new solid-phase radioimmunoassay was applied to tetanus toxoid antibodies of the booster response. Total concentrations varied from 1700-26 000 L units/ml (13-200 micrograms/ml). Concentrations of IgG1 antibodies were 1600-25 000 units/ml (average 91% of the total) and concentrations of IgG4 antibodies 12-6900 units/ml (average 6.9% of the total). Antibodies of the other four isotypes were not detected in all sera and together they never exceeded 3% of the total.
Asunto(s)
Anticuerpos Antibacterianos/análisis , Alotipos de Inmunoglobulinas/análisis , Toxoide Tetánico/inmunología , Anticuerpos Antibacterianos/inmunología , Humanos , Masculino , Radioinmunoensayo/métodos , Yersinia/inmunologíaRESUMEN
BACKGROUND & AIMS: Oxygen free radicals, generated by xanthine oxidase (XO) and activated leukocytes, are involved in reperfusion injury in experimental liver transplantation. The roles of XO and neutrophil activation during reperfusion in clinical liver transplantation were studied. METHODS: In 10 patients undergoing liver transplantation, we assessed plasma concentrations of circulating XO by enzyme-linked immunosorbent assay (ELISA), the purine metabolites hypoxanthine, xanthine, and urate by high-performance liquid chromatography, lactoferrin by ELISA, and malondialdehyde fluorometrically up to 48 hours postoperatively. RESULTS: During reperfusion after portal vein declamping, elevated plasma concentrations of XO (52.1 ng/mL [range, 8.0-440.1]), hypoxanthine (81.62 micromol/L [48.2-108.7]), xanthine (21.01 micromol/L [8.7-22.3]), and lactoferrin (532.6 ng/mL [370.4-1326.6]) were observed compared with the preoperative levels (0 ng/mL [0-12], 1.88 micromol/L [0.62-3.15], 0.95 micromol/L [0-0.41], and 164.3 ng/mL [73.7-334.1], respectively; all P < 0.05). No changes occurred in urate or malondialdehyde. After portal vein declamping, XO, hypoxanthine, and xanthine levels were substantially greater in the hepatic than portal vein (all P < 0.05). Marginal transhepatic differences occurred in lactoferrin. CONCLUSIONS: Reperfusion during liver transplantation is associated with liberation of xanthine oxidase, hypoxanthine, and xanthine from the liver into the circulation. During reperfusion, intravascular neutrophil activation takes place in the hepatic circulation.
Asunto(s)
Trasplante de Hígado , Activación Neutrófila/fisiología , Xantina Oxidasa/sangre , Adulto , Cromatografía Líquida de Alta Presión , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Hipoxantina/sangre , Periodo Intraoperatorio , Lactoferrina/sangre , Circulación Hepática/fisiología , Masculino , Persona de Mediana Edad , Periodo Posoperatorio , Reperfusión , Ácido Úrico/sangre , Xantina/sangreRESUMEN
Respiratory distress syndrome (RDS) and development of bronchopulmonary dysplasia (BPD) are characterized by endothelial cell damage. Persistent pulmonary hypertension of the newborn (PPHN) is a disorder that alters the pulmonary microvasculature. Immunohistochemistry for VEGFA(165), an endothelial cell mitogen, and its receptor Flt-1, was performed on lung tissues from autopsies from four fetuses, three preterm infants, four term infants without primary lung disease, four infants with BPD, and four infants with PPHN. VEGF was measured in tracheal aspirates from 31 preterm infants, 5 intubated term infants without primary lung injury, and 12 infants with PPHN during the first 10 postnatal days, and from 8 infants with BPD. Immunohistochemistry for VEGF and Flt-1 was similar in fetuses, preterm infants, and term infants: for VEGF mostly in bronchial epithelium and alveolar macrophages, and for Flt-1 mostly in vascular endothelial cells and bronchial epithelial cells. In patients with BPD, and PPHN, staining for VEGF and Flt-1 appeared also in Type II pneumocytes. Preterm infants with more severe RDS had lower VEGF than those who recovered. The persistent expression of VEGF and Flt-1 during the fetal and neonatal period supports a physiological role for VEGF in human lung development. The lower pulmonary VEGF in preterm infants with more severe RDS may contribute to the pathophysiology of the acute lung injury. In BPD, the expression of VEGF in alveolar epithelium may represent a compensatory increase after the acute phase of the lung disease. In PPHN, that more cell types express VEGF and Flt-1, and the tendency toward a higher concentration of pulmonary VEGF may represent enhanced production of VEGF in response to impaired endothelial function.